D E V E LO P M EN T O F A N E W U P L C®/ M S M E T HO D FO R S YS T EMAT IC T OX ICO LOGIC A L A NA LYSIS

Robert Lee1, Mark Roberts1, Alexandre Paccou2 and Michelle Wood1 1Waters Corporation, Manchester, UK. 2Waters Europe, Paris, France.

ABST RACT

A method has previously been described for the systematic

toxicological analysis (STA) of biological specimens. This method

comprised a 26 minute HPLC separation, in combination with the

collection of full scan mass spectral data and has been success-

fully applied for the analysis of routine samples in laboratories

worldwide over the last 5 years.

Since this method was first described, there have been some

significant advances in the available technology. In 2004, the

revolutionary separation technique, UPLC® was introduced.

We now present our latest STA method. This technique exploits the

rapid separation afforded by UPLC combined with the ultra-fast

scanning capabilities of the Waters TQ Detector, providing a com-

prehensive analysis in only 15 minutes – a time-saving of 40 %.

INT RODUCT ION

Broad screening techniques are routinely applied to biological

samples for the identification of toxicants. In the field of forensic

toxicology, the analysis of ante and postmortem specimens may

be necessary to investigate cases of alleged chemical submission,

to identify the use of illicit compounds and to ascertain the cause

of death. In emergency toxicology, analysis may be required for

the investigation of accidental poisoning, suspected overdose or

following an adverse reaction to prescriptive or over-the-counter

medication. In these latter situations, in particular, analytical

speed and assay turnaround time can be a critical element.

Previously, we have described a screening method based on LC-MS1.

The method comprised chromatographic separation (26 minutes)

combined with full scan detection. Resultant data were collected

and matched against a spectral database which had been created

under identical analytical conditions.

T he database contained information for approximately

500 toxicologically-relevant analytes. Since its release more than

4 years ago, this method has been successfully used in laboratories

worldwide.

One of the main challenges facing forensic laboratories these days,

is a need to increase service whilst holding costs to a minimum. The

laboratory can now play a major role by providing greater sample

throughput and expanding analytical capability whilst maintaining,

or if possible improving, the data quality.

We present our latest STA method which utilises the newest state-

of-the-art LC and MS technologies.

INNOVAT IV E T ECHNOLOGIES

ACQUITY UPLC 2004 saw the advent of UltraPerformance® LC

(UPLC); a major breakthrough in separation science which has

provided scientists, from all disciplines, with vast improvements

over their traditional HPLC techniques. The smaller particle

size (sub-2 µm) of the UPLC columns (Figure 1) leads to enhanced

chromatographic peak resolution; sharper and narrower peaks with

increased signal to noise.

Figure 1. The Waters ACQUITY TQD system and ACQUITY UPLC column featuring eCordTM technology. The eCord electronically stores all the information for full traceability of your experiments including; date of column installation, certificate of analysis, number of injections, maximum temperature and pressure - a full column history.

This novel technique also allows a dramatic reduction of the sample

run time. These enhancements ultimately result in the provision

of superior analyte detection combined with increased sample

throughput.

The Waters TQ Detector

UPLC systems can generate peak widths as narrow as one second

at half-height. Consequently, this can pose a significant challenge

for peak detection. To fully exploit the increased analytical

capabilities afforded by UPLC, an appropriate detection system

is also required. This system needs to have a sampling rate high

enough to provide sufficient definition of the chromatographic

peaks to allow reproducible detection and integration. The Waters

TQ Detector has been designed to provide higher speed data

acquisition whilst maintaining data quality with a maximum MS

scan-speed of 10,000 amu/s. Ultra-fast polarity switching in only

20 ms means that both positive and negative ionising compounds

can be detected in the same run.

OV ERV IEW OF SC REENING MET HODOLOGY

UPLC/MS Library

I. Library Concept

This latest library method utilises the same library concept as

previously described by Humbert1 i.e., for each analyte, mass

spectra are collected under multiple fragmentation conditions.

The degree of fragmentation is controlled by varying the cone

voltage in the source of the mass spectrometer.

This process, known as in-source collision-induced dissociation

(in-source CID), can be performed simultaneously in both ES+

and ES- modes, hence library entries can be created for positive

and negatively ionising compounds (Figure 2). Retention time

(RT) information is also recorded for each analyte which provides

additional confidence in the result. Data for authentic samples are

collected under exactly the same UPLC/MS conditions as those

used for library creation.

II. Library content

A new database has been created and contains data for 500 of the

most commonly-encountered toxicants including illicit drugs and

metabolites, and prescribed drugs.

Figure 2. Fragmentation patterns for benzoylecgonine in positive ionisation mode (A) and salicylic acid in negative ionisation mode (B). Only spectral data acquired at 20, 50 and 80 V are shown for simplicity. However, typically the library contains 6 mass spectra (recorded at 6 cone voltages) for each analyte, in addition to RT.

80 V

50 V

20 V

Incr

easin

g fra

gmen

tatio

n

(A) (B)

[MH] + [MH] -

The library constitutes a powerful and reliable tool for the

toxicology laboratory. It has been extensively investigated

for accuracy of RT and spectral data within both Waters and

collaborator’s laboratories. The library is also easy to maintain

and fully appendable by the user.

III. Utility of full scan data

The collection of full scan MS data provides a more comprehensive

screening for true unknowns than any targeted LC-MS/MS

approach. The result is a more complete (rather than a targeted/

restricted) dataset. As the acquired data files remain unaltered,

the data may be interrogated retrospectively if required; this can

be performed even without the need to re-analyse the sample.

The flexibility of the Waters TQ Detector allows the user to collect

full scan data for broad screening but also to make use of the

L C - M S / MS capabilities to confirm the presence of proposed

analytes without the need for additional instrumentation.

Confirmation assays are typically performed by using the

instrument in multiple reaction monitoring (MRM) mode

and require the ion ratio of qualifier and quantifier ions to be

determined.

EXPERIMENTAL

LC conditions

LC System: Waters ACQUITY UPLC® System

Column: ACQUITY UPLC® HSS C18 Column

2.1 x 150 mm, 1.8 µm

Column Temp: 50 ˚C

Flow Rate: 400 µL/min.

Mobile Phase A: 5 mM ammonium formate, pH 3.0

Mobile Phase B: Acetonitrile with 0.1 % formic acid

Initial Conditions: 87 % Mobile Phase A

Gradient: Gradient increasing to 95 % Mobile Phase B

Analysis Time: 15 Minutes

Weak wash: 10 % acetonitrile in water (600 µL)

Strong wash: 95 % acetonitrile in water (200 µL)

MS conditions

MS System: Waters TQ Detector

Capillary Voltage: 3.5 kV

Cone Voltage: 20 V to 95 V (in 15 V increments)

Desolvation Temp: 400 ˚C

Desolvation Gas: 800 L/Hr

Source Temp: 150 ˚C

Acquisition Range: m/z 80—650

Software

Waters MassLynx™ software v4.1 was used for data acquisition

and the ChromaLynx™ application manager2 was used for data

processing. ChromaLynx is a unique data processing software

based on deconvolution techniques.

The application manager automatically examines the chromatograms

produced at each cone voltage, detects the components and

calculates the average spectral match factor (MF) against the

library (maximum MF = 1000). Candidates are assigned with the

following symbols according to the total accuracy of the match:

These are user-definable criteria (typically MF >700, 500-700

and <500 respectively, are utilised).

X?

RESULTS AND DISCUSSION

The minimised volumes and optimised flow paths of the UPLC

instrumentation allow a precise and rapid delivery of mobile phase

gradients and column equilibration. The total analytical time for

the new STA method has been reduced from 26 min to 15 min

as seen in Figure 3.

The increased speed and resolution associated with UPLC results

in a significant reduction in peak width. Figure 4 shows an example

of the analysis of colchicine; peak widths (half-height) are reduced

from 8.4 seconds with HPLC to 2.1 seconds with UPLC.

Such narrow peaks would pose a potential problem for any

‘normal’ MS detector and could compromise data quality by

producing insufficient or poor reproducibility of spectral data for a

qualitative analyses and even poor sensitivity and reproducibility

for quantitative analyses. In this STA method, scans rates

of >7000 amu/sec are used. The figure below demonstrates that

when coupled to the ultrafast scanning Waters TQ Detector, data

quantity and quality is maintained.

Figure 4. Analysis of colchicine. The fast scanning capability of the TQ Detector ensures that a sufficient number of scans is maintained (11 scans in each case), even though UPLC separation leads to much narrower, peaks i.e., peak width (at half height) for colchicine has been reduced from 8.4 sec (HPLC) to 2.1 sec (UPLC).

8.4 sec

2.1 sec

RT 5.97

RT 13.3

Figure 3. Extracted ion chromatograms of a mixture of standards analysed using the original screening configuration i.e., Alliance® 2695 plus Quattro micro™ (top trace) versus the latest instrumentation i.e., ACQUITY TQD system (bottom trace). Total analysis time has been reduced from 26 to 15 min.

0 5 10 15 20 25 min

A

B

C

D

E

F

0 5 10 15 20 25 min

A

B

C

D

EF

A: NADOLOL

B: LSD

C: AMOXAPINE

D:TRIMIPRAMINE

E: DESALKYLFLURAZEPAM

F: PRAZEPAM

Sharper chromatographic peaks typically leads to increased

signal to noise ratios and consequently improved detection

limits. The increased chromatographic resolution also provides

enhanced deconvolution of the data and peak identification by the

ChromaLynx™ application manager.

Figure 5 shows a typical results browser. The data was obtained

following the analysis of an authentic urine sample. The sample

was prepared using liquid:liquid extraction (LLE) prior to analysis

by the STA method. Several compounds and metabolites were

identified. The candidate listing includes the name of the

proposed compound followed by the observed retention time (RT),

the reference/library RT (within the parentheses) and the average

match factor.

The results viewed in the browser can be reported using the report

generator option. An example of one available report format is

shown in Figure 6.

(i) Chromatogram TIC

(iii) Spectrum view

(ii) Candidate listing

Figure 5. Analysis of an authentic urine sample. The browser shows; (i) the total ion chromatogram (TIC); (ii) the list of proposed candidates; (iii) the spectral match for function 5 (cone voltage 65 V) of one of the proposed candidates (EDDP). The spectrum view window allows a direct visual comparison of the acquired spectral data with the library data. In this example an excellent average MF was observed i.e., 924 out of a possible max. 1000.

Figure 6. Example of a simplified report for a serum sample containing clozapine and its metabolite desmethylclozapine. Clozapine was the top hit (match) in all of the 6 cone voltage functions examined. The metabolite was the top hit in 5.Both showed excellent average MFs against the library.

CONCLUSIONS

Toxicology laboratories require the ability to perform STA to

screen and identify unknown compounds in a variety of complex

biological specimens. In addition, these laboratories face an

increased demand in sample throughput and the need to analyse

a greater number of samples in a shorter time.

The superior speed and resolution afforded by the use of UPLC,

combined with the ability of the TQ Detector to match UPLC

performance with rapid polarity switching and ultra-fast scanning,

ensure the laboratory can perform prompt, efficient and thorough

analyses.

The method described in this application note utilises full scan

spectra and retention time to identify toxicants. Analytical

time is just 15 minutes thus maximising sample throughput and

optimising workflows. The comprehensive features of ChromaLynx™

deconvolution and automatic data processing software ensure

that the maximum number of possible compounds are detected,

identified and reported.

A starter project (including library) is provided which contains

everything the user needs to perform a comprehensive screen.

The methods are supplied on DVD and are ready for immediate

implementation within the laboratory with minimal user

intervention. The DVD also contains supporting documentation and

literature including a user manual and a ‘step by step’ workflow

specifically designed with the new user in mind; a simple guide

from initial instrument setup (including system verification using

a system suitability mixture) through to the analysis of authentic

samples.

A dedicated team of Waters applications specialists are also

available worldwide to implement and provide training.

The ability to add additional compounds to the already

comprehensive library, in combination with retrospective

analytical capabilities, ensure that this STA method will continue

to remain versatile and relevant for the future.

Figure 7. Positive identification of dosulepine in addition to several other toxicological compounds in a forensic sample. Dosulepine was not included in the routine targeted LC-MS/MS-based screen and therefore was not initially identified. Methyl-clonazepam was added to the sample prior to analysis and used as an internal standard to verify chromatographic performance.

2 4 6 8 10 12 14 min

%

0

100 7.537.41

3.43

1.05

0.72 1.26 2.62

6.234.83

4.04 5.33 7.19

10 64

9.85

9.579.04

8.51

11.76

1 .83

.

2

©2009 Waters Corporation. Waters, ACQUITY UPLC, UltraPerformance LC and UPLC are registered trademarks of Waters Corporation and The Science of What’s Possible are trademarks of Waters Corporation. All other trademarks are the property of their respective owners.

February 2009 72002905EN KK-PDF

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ACKNOWLEDGEMENTS

Special thanks to Luc Humbert, Camille Richeval and Michel

Lhermitte of the CHRU, Lille, France for their considerable scientific

contributions.

REFERENCES

1. General Unknown Screening for Drugs in Biological Samples by LC/MS. Luc Humbert, Michel Lhermitte, Frederic Grisel. Waters application note 720001552EN

2. MassLynx 4.1 Brochure. Waters brochure reference: 720001408EN

3. Targeted MRM Screening for Toxicants in Biological Samples by UPLC-MS/MS. Mark Roberts, Robert Lee and Michelle Wood. Waters application note 720002749EN.